Little is known about the mechanisms involved in the cellular uptake of folates. There is very strong evidence for the existence of two transport systems; one mediating the transport of oxidized folates, and another for reduced folates and amethopterin (methotrexate). We have recently been able to elucidate early steps in the uptake of oxidized folates. To this end we have purified to apparent homogeneity a particulate folate binding protein (receptor) from human placenta, also present in the plasma membrane of human erythrocytes, which appears to mediate the uptake of oxidized folates. A variety of tissues contain both particulate and solube folate binding proteins (FBP) whose role in mediating oxidized folate uptake has been poorly understood. We have also recently purified to apparent homogeneity, characterized and compared the particulate and soluble FBPs from human milk. Our studies show that these proteins are closely related with regard to their kinetics of folate binding, affinity for various folate analogues, amino acid and carbohydrate composition and antigenic determinants. In addition they bear a close biochemical relationship with the human placental folate receptor. The particulate FBP, however, has more hydrophobic amino acids and consequently binds more Triton X-100 than the soluble FBP, a finding which is consistent with its membrane origin and relationship with the placental folate receptor. We plan to determine if a separate receptor for the uptake of reduced folates exists in human tissues. Furthermore if such a receptor is present, we plan to purify, characterize and compare it with the purified components of the oxidized folate transport system. With the antibodies we have already obtained against the purified human placental folate receptor, the particulate and soluble human milk FBPs as well as antibodies we will attempt to obtain against the putative reduced folate receptor, we will develop specific radiommunoassays for each of these proteins. Subsequently, using these antibodies and specific radioimmunoassays we will study various aspects of receptor regulation, specificity of these receptors in mediating folate and folate analogue uptake and alterations of receptor in folate transport defective states. These studies will form the basis for our understanding of the mechanisms involved in regulation of folate uptake in normal cells as well as folate transport mutant cells.